This R21 exploratory proposal uses an integrated target-oriented approach in the rational development of drugs that may effectively shorten the duration of therapy in tuberculosis and/or improve the treatment of multi-drug resistant TB. There exists a broad consensus in the TB research community that the key to shortening the duration of drug therapy without an increase in relapse rates lies in the specific targeting of proteins that are essential to the non-replicating persistence (NRP) of Mycobacterium tuberculosis. One such target is malate synthase (MS). It has been shown that MS is crucial for persistent growth in mice and is over-expressed in vitro models of NRP. Recent success in the structure elucidation of the MS protein and the availability of functional assays for MS opens a way to apply computer-aided drug design methods in concert with medicinal chemistry efforts to discover new therapeutic agents for NRP TB. We hypothesize that it would be possible to identify and design inhibitors for MS using a combination of computer-aided drug design and medicinal chemistry. Those compounds demonstrating activity at low micromolar range against MS will be screened at the whole cell level for the ability to kill replicating and NRP M. tuberculosis in a microplate assay based on the Wayne low oxygen model and for selectivity with respect to mammalian cell toxicity. This iterative process is expected to yield several low micromolar, lead- or drug-like inhibitors of MS. Such leads would be candidates for further development including more extensive medicinal chemistry efforts and the evaluation in animal models of NRP in subsequent projects. To achieve this goal, our specific aims are as follow: (1) Identify lead- or drug-like low molecular weight inhibitors of MS using a search for close analogs of the natural substrates of MS and identification of new inhibitors using de novo/rational drug design, in silico screening of virtual focused combinatorial libraries and the databases of commercially available chemical compounds;to synthesize the best candidates;(2) Assay candidate drugs for their inhibition of MS functional activity. (3) Identify compounds demonstrating significant selective activity against actively growing and/or non-replicating, persistent (NRP) Mycobacterium tuberculosis. Prioritize on the basis of activity, cytotoxicity and resultant selectivity indices. PUBLIC HEALTH RELEVANCE: This proposal is intended to find new malate synthase inhibitors, a potential new target for non- replicating persistent tuberculosis. This project brings together for the first time an NRP-associated target, malate synthase, libraries of compounds with properties that optimize the chances of finding useful lead compounds, computer-aided drug design approach, and medicinal chemistry.